Dihydropyridines (DHPs) are a class of organic molecules which regulate voltage-dependent calcium channels in a variety of tissues. In skeletal muscle the DHP binding proteins (DHPBP) play an important role in DHPBP and its individual subunits to help to define the role of this oligomeric protein in E-C coupling. The DHPBP of rabbit skeletal muscle is a oligomeric protein with a 170K (alpha 1), a 140K (alpha 2), a 52K (beta), a 35K (gamma), and a 30K (delta) subunit. A small fraction of the alpha 1 subunit also exists as a non-proteolyzed 212K polypeptide. Definition of the arrangement of these subunits in the membrane and mapping of the location of physiologically significant drug binding sites on the subunits will greatly advance our understanding of how this protein functions as both an ion channel and a voltage sensor. We propose to prepare monoclonal antibodies to peptides and to subunits to use to: 1) establish the topology of the individual subunits with respect to the membrane by a combination of epitope mapping and electron microscopy, 2) determine the relative locations of the subunits with respect one another by analyzing the composition of partially dissociated and/or reconstituted complexes, 3) purify individual subunits of the DHPBP, and 4) probe for effects on dihydropyridine binding and on calcium channel activity. We also propose to determine the location of pharmacologically important binding sites, and the disulfide bond between alpha 2, and delta by peptide mapping and N-terminal amino acid sequencing.